摘要
目的:构建含荧光素酶(luciferase,luc)基因的短发夹状RNA(short hairpin RNA,shRNA)表达质粒,并观察其在肝癌细胞Bel-7402中抑制外源荧光素酶的表达. 方法:设计的两条反向重复的多聚核苷酸序列,退火形成双链DNA,再与双酶切后的载体PGE-1连接,构建pshRNA-luc重组质粒,与荧光素酶基因表达质粒pCMV- luc共转染肝癌细胞株BEL-7402,检测其对荧光素酶表达的影响. 结果:PCR和DNA序列分析证实了重组质粒构建成功. pshRNA-luc对共转染的pCMV-luc中的荧光素酶具有明显的抑制作用,抑制率可达86%(P<0.01). 结论:构建的pshRNA-luc表达质粒能有效地抑制荧光素酶在肝癌细胞中的表达,为RNA干扰用于肿瘤的基因治疗打下基础.
AIM: To construct a recombinant plasmid containing short hairpin RNA of luciferase in order to suppress the expression of luciferase gene in hepatocellular carcinoma cells line Bel-7402. METHODS: Two inverted repeating oligonucleotides, double-stranded DNA which dropped temperature were designed, and then cloned into PGE-1 vector digesting by the double restricted endoenzymes to generate the plas-mids pshRNA-luc, pshRNA-luc and pCMV-luc, which were cotransfected into hepatocellular carcinoma cells line Bel-7402 to detect effect of luciferase expression. RESULTS: The size of the PCR product was 652 bp. DNA sequencing showed the sequence of recombinant plas-mids pshRNA-luc was successfully constructed. The recombinant plasmids suppressed the expression of luciferase gene by 86% in Bel-7402 cells (P<0.01). CONCLUSION: Short hairpin RNA of luciferase can efficiently suppress its expression in hepatocellular carcinoma cells Bel-7402.
出处
《世界华人消化杂志》
CAS
2004年第9期2045-2048,共4页
World Chinese Journal of Digestology
基金
湖北省卫生厅资助项目
No.JX1B116~~
